Influenza A virus is a member of the orthomyxoviridae virus family of (−)-sense RNA viruses. The Influenza A viral genome is composed of 8 segments or chromosomes which encode 11 proteins. During infection, these (−)-stranded RNAs are converted to both (+) strand messenger RNAs and a set of full length complementary genomic RNAs (or cRNAs) which serve as templates for genomic replication by a virus-encoded RNA-dependent RNA polymerase. Viral proteins expressed from the (+) strand messenger RNAs go about the task of establishing infection and facilitating viral replication, a process which ends in the amplification, assembly, and logarithmic release of virus particles containing the initial 8 (−) strand chromosomes.
The processes associated with the transcription and replication of the influenza A genome have been under investigation for decades. All eight chromosomes of every influenza A strain (including H1N1 seasonal, H1N1 “swine”, H3N2, and H5N1 “avian”) contain identical 5′ and nearly identical 3′ untranslated regions (UTRs) flanking the protein-coding portion of the sequence which otherwise encode distinct proteins and strain-specific variants. Experimental results demonstrate that the UTRs are recognized by the viral RNA-dependent RNA polymerase (vPol) as a promoter element and highlight the importance of the UTR sequences in viral gene expression and replication. Hence, the viral polymerase and its cognate ligand control the viral life cycle and are critical targets for therapeutic intervention.
Due to the partial complementarity of the UTR sequences to each other, different models for the UTR structure recognized by vPol have been proposed, including the panhandle, RNA fork, and corkscrew conformations. Although the structure formed by the UTRs is probably dynamic, the model most likely to represent the actual structure of the UTRs and hence the promoter element for vPol-driven gene expression is the corkscrew model. The adoption of this highly unusual tetrahelical (also referred to as “corkscrew”- or “panhandle”-like) structure by the UTRs is supported by at least two different lines of evidence provided by genetic and solution NMR studies. For the former, genetic point mutants spanning the entire length of the two UTRs were created and assessed in their promoter activity and the resulting gene expression data were consistent with the corkscrew conformation model. Further studies using NMR spectroscopy solved the solution structure of a synthetic RNA possessing correctly oriented UTR sequences and again determined a corkscrew-like structure. Biochemical and genetic assays were also able to delineate the critical promoter sequences recognized by vPol as residing between nucleotides 9-12 of the UTR with the “bulge” structure in the vicinity of A11 as a central player in recognition and polyadenylation.
Further studies have provided insights to the functionality of the panhandle structure (see e.g., JBC (2011) 286, No. 26, pp. 22965-22970; NAR (1999), 27, No. 5, pp. 1392-1397). However, these insights have failed to provide a rational design approach to postulate and identify an inhibitor that would bind to the panhandle structure and thereby reduce or stop viral propagation. Only recently (see Biol. Pharm. Bull. (2013) 36(7) 1152-1158), certain pyrrole-imidazole polyamides were described as being able to bind to DNA and RNA double stranded structures. Unfortunately, the pyrrole-imidazole (PI) polyamides used showed only moderate affinity to the Influenza A panhandle structure in vitro and were not tested in vivo for any antiviral activity. All publications identified herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
More recently, high throughput screening has identified several compounds with significant antiviral activity against an apparently conserved viral target that is located within the viral replication and/or gene expression machinery, most likely the viral RNA-dependent RNA polymerase as described in U.S. Pat. No. 8,633,198. While these compounds have exhibited promising in vitro and in vivo activity, several drawbacks still remain. Among other things, where such compounds bind to a viral protein, resistance is likely to develop unless these compounds target an essential and highly conserved structure in the polypeptide.
Thus, despite the relatively detailed knowledge of the promoter structure and life cycle of influenza viruses, drug development for inhibitors of viral replication has not yielded the desired therapeutically effective compounds that target the viral UTR sequences that appear to be essential for viral replication and protein expression. Moreover, there is also a lack of rational drug design approaches for viral inhibitors that is independent of first-pass in vitro high throughput screening. Therefore, there is still a need to provide improved antiviral compositions and methods, and especially those that are specific for seasonal, pandemic, and emerging influenza viruses.